1. Field of the Invention
This invention relates to a process for recovering a carboxylic acid made by the biological oxidation of a substrate by a microorganism from an aqueous media such as a fermentation broth.
2. Description of Related Art
Standard methods for recovering carboxylic acids in general and polycarboxylic acids in particular from fermentation broths are typically based on the physical separation of the spent microorganism cells from the aqueous phase such as by centrifugation followed by precipitation of the carboxylic acid as a result of pH reduction of the aqueous phase. This method is unsatisfactory for a number of reasons, the most notable of which includes the problem of physically separating the spent cells and then acidifying the cell-free broth to effect the precipitation of the carboxylic acid. The precipitation of the carboxylic acid is time consuming and the separation and isolation of the precipitated carboxylic acid is not always clean, i.e., there can be impurities which adversely affect the quality and purity of the final product.
Accordingly, there is a continuing need for improved processes for recovering carboxylic acids from a fermentation broth.
The present invention involves an improved process for the recovery of a carboxylic acid made by the biological oxidation of a substrate by a microorganism such as a yeast. Carboxylic acids are recovered from a fermentation broth by extracting the broth with a solvent containing one or more olefins without the need for first removing the spent microorganism cells. In one aspect, a cosolvent is combined with one or more olefins to create a solvent which favorably adjusts the partition coefficient. A preferred solvent for the extraction of carboxylic acids from a fermentation broth contains a mixture of tertiary-butyl acetate and diisobutylene.
In another aspect, a method for isolating at least one carboxylic acid from an aqueous mixture is provided which includes providing an aqueous mixture containing at least one carboxylic acid, contacting a solvent containing at least one olefin with the aqueous mixture, allowing the at least one carboxylic acid to separate into a solvent and isolating the solvent containing the at least one carboxylic acid from the aqueous mixture. A co-solvent may be combined with the at least one olefin.
Solvents are provided, which in one embodiment, include a mixture of a minority amount of tertiary butyl acetate and a majority amount of diisobutylene. During the process of recovering the carboxylic acids, one or more co-solvents may be included which generally aid in the process, e.g., by reducing the amount of solvent needed to remove the carboxylic acids, and/or the number of extraction cycles necessary to adequately remove the carboxylic acids from the fermentation broth.
After employing a fermentation procedure to produce a carboxylic acid, the viscosity of the fermentation broth may optionally be adjusted, preferably by heating the fermentation broth, to form a flowable liquid. The pH of a fermentation broth which contains one or more carboxylic acids may be optionally adjusted to a value of from at least about 1.5 to about 7.0. The carboxylic acid containing broth is contacted with a solvent containing one or more olefins to extract the carboxylic acid. The carboxylic acid may then be isolated by separating crystals of the carboxylic acid from the extraction solvent.
Except in the claims and the operating examples, or where otherwise expressly indicated, all numerical quantities in this description indicating amounts of material or conditions of reaction and/or use are to be understood as modified by the word xe2x80x9caboutxe2x80x9d in describing the broadest scope of the invention. Practice within the numerical limits stated is generally preferred. Also, throughout this description, unless expressly stated to the contrary: percent, xe2x80x9cpartsxe2x80x9d of, and ratio values are by weight; the description of a group or class of materials as suitable or preferred for a given purpose in connection with the invention implies that mixtures of any two or more of the members of the group or class are equally suitable or preferred; description of constituents in chemical terms refers to the constituents at the time of addition to any combination specified in the description or of generation in situ by chemical reactions specified in the description, and does not necessarily preclude other chemical interactions among the constituents of a mixture once mixed; and the term xe2x80x9cmolexe2x80x9d and its grammatical variations may be applied to elemental, ionic, and any other chemical species defined by number and type of atoms present, as well as to compounds with well defined molecules.
It is understood that a carboxylic acid is any compound containing one or more carboxyl groups. A polycarboxylic acid is any compound having two or more carboxyl groups.
A flowable liquid is a fluid whose molecules are free to move past one another while remaining in sliding contact.
The process for the recovery of a carboxylic acid according to the invention was based on fermenting a microorganism in a culture medium which is comprised of a nitrogen source, and at least an organic substrate and then recovering the carboxylic acid by contacting the broth with a suitable extractant. The organic substrate can be any compound which can be oxidized to a compound having at least one carboxyl group by biooxidation. For the production of carboxylic acids, the substrate can be any compound having at least one methyl group, a terminal carboxyl group and/or a terminal functional group which is oxidizable to a carboxyl group by biooxidation. The substrate can also contain one or more carbonxe2x80x94carbon multiple bonds and/or one or more carboxylic or heterocyclic aromatic rings. The microorganism can be any microorganism that is capable of biologically oxidizing an organic substrate as set forth above to a compound having at least one carboxyl group.
The process for the recovery of a carboxylic acid is applicable to the production by fermentation of any carboxylic acid that has between 3 and 36 carbon atoms, preferably 5 to 36 atoms and more preferably 9 to 36 carbon atoms. The process for the recovery of a carboxylic acid is particularly applicable to the production of polycarboxylic acids by fermentation and most particularly to the production of dicarboxylic acids. Examples of such dicarboxylic acids include, but are not limited to, oxalic acid, maleic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, phthalic acid, azelaic acid, sebacic acid, dodecanedioic acid, brassylic acid, 9-octadecenedioic acid, C-36 dimer acid and isomers thereof. The process is also applicable to the recovery of monobasic carboxylic acids which can be saturated, unsaturated or polyunsaturated. Examples of such monocarboxylic acids include, but are not limited to: caprylic, pelargonic, capric, undecylic, lauric, myristic, pentadecanoic, palmitic, heptadecanoic, stearic, arachidic, palmitoleic, oleic, erucic, linoleic, and linolenic and isomers thereof.
The microorganisms can be any microorganism capable of biooxidizing the substrate as defined herein. Typically, such a microorganism will be a yeast. Several strains of yeast are known to excrete alpha, omega-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids as the carbon source. Certain strains are set forth, e.g., in U.S. Pat. No. 5,254,466 and PCT/US99/20797, the entire contents of which are each incorporated herein by reference. Preferably, the microorganism is a beta-oxidation blocked C. tropicalis cell which has been genetically modified so that the chromosomal POX4A, POX4B and both POX5 genes have been disrupted. The substrate flow in this strain is redirected to the omega-oxidation pathway as the result of functional inactivation of the competing xcex2-oxidation pathway by POX gene disruption. The strain may also have one or more reductase genes amplified which results in an increase in the amount of rate-limiting omega-hydroxylase through P450 gene amplification and an increase in the rate of substrate flow through the xcfx89-oxidation pathway. Such strains are discussed in detail in PCT/US99/20797.
The process for making a dicarboxylic acid includes fermenting a suitable microorganism in a culture medium comprised of a nitrogen source, an organic substrate and a cosubstrate wherein the substrate is a compound having one carboxyl group and one methyl group or is a compound having one methyl group and a functional group that can be at least partially hydrolyzed to a carboxyl group and optionally wherein the substrate is partially neutralized. Saponification may be desirable for some raw materials.
Examples of nitrogen sources are disclosed in U.S. Pat. No. 5,254,466 and PCT/US99/20797. The cosubstrate can be any fermentable carbohydrate such as glucose, fructose, maltose, glycerol and sodium acetate. The preferred cosubstrate is glucose, preferably a liquid glucose syrup, for example, 95% dextrose-equivalent syrup, or even lower dextrose-equivalent syrups. Such materials contain small amounts of disaccharides, trisaccharides, and polysaccharides which can be hydrolyzed during the fermentation by the addition of an amylase enzyme.
The organic substrate can be any compound having at least one methyl group which can be bioxidized. One type of organic substrate includes alkanes having from 3 to 36 carbon atoms, preferably those having 9 to 36 carbons, examples of which include, but are not limited to, nonane, decane, undecane, dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecane, octadecane, nonadecane, eicosane and their isomers. The organic substrate can also be any saturated aliphatic compound having at least one terminal methyl group, a terminal carboxyl group and/or a terminal functional group which is oxidizable to a carboxyl group by biooxidation. The organic substrate can also be any unsaturated aliphatic compound having at least one internal carbonxe2x80x94carbon double bond and at least one terminal methyl group, a terminal carboxyl group and/or a terminal functional group which is oxidizable to a carboxyl group by biooxidation. One source of organic substrates is high oleic acid sunflower fatty acids (HOSFA) which is commercially available from Cognis Corp., Cincinnati, Ohio.
In those instances where the process according to the invention is applied to the production of dicarboxylic acids, the organic substrate is preferably any compound having one carboxyl group and one methyl group or is a compound having one methyl group and a functional group that can be at least partially hydrolyzed to a carboxyl group. Thus, the organic substrate in this case can be any aliphatic saturated or unsaturated monocarboxylic acid except formic acid and acrylic acid. Examples of carboxylic acid substrates include, but are not limited to: caprylic, pelargonic, capric, undecylic, lauric, myristic, pentadecanoic, palmitic, heptadecanoic, stearic, arachidic, palmitoleic, oleic, erucic, linoleic, and linolenic and isomers thereof. The organic substrate can also be an aromatic monocarboxylic acid having a methyl group, the simplest example of which is o, m or p-methyl benzoic acid.
The substrate can be optionally partially neutralized with a base, preferably an alkaline earth metal hydroxide prior to the addition of the substrate to the fermentation broth. Certain preferred hydroxides are calcium, magnesium, sodium and potassium hydroxide. While the organic substrate will preferably be a monocarboxylic acid for the production of polycarboxylic acids, it can be any compound having one carboxyl group and one methyl group or having one methyl group and a functional group thereby permitting at least partial neutralization of the carboxyl group formed in the hydrolysis. Particularly preferred monocarboxylic acids are oleic acid and pelargonic acid.
A process for the recovery of a carboxylic acid according to the invention involves optionally adjusting the viscosity of the broth preferably by heating the fermentation broth to form a flowable liquid. Adjusting the viscosity of the broth allows for better contact between the broth and the solvent containing one or more olefins that will be used for the extraction. This improves the effectiveness of the extraction. The preferred temperature of the broth is from about 0xc2x0 C. to about 100xc2x0 C. and more preferably from about 70xc2x0 C. to about 80xc2x0 C.
A preferred embodiment incorporates a pH of the fermentation broth in a slightly acidic pH range. An optional step of the process for the recovery of carboxylic acids following the fermentation process is the adjustment of the pH of the fermentation broth in the range from about at least 1.5 to about 7.0, preferably in the range from about 3.0 to about 6.0, and more preferably in the range from about 4.5 to about 5.5 and most preferably about 4.7. Typically, the pH value of the broth will fall in the range of from 5.0 to 7.5 but may be higher than 7.5 depending upon the fermentation conditions, the nature of the substrate, cosubstrate, the microorganism and the carboxylic acid formed in the fermentation. The pH adjustment takes place during or after the fermentation process. Typically, carboxylic acids recovered by the process are unsubstituted aliphatic carboxylic acids. These acids will normally have a pKa in the range of from about 4 to about 5. The acid used to adjust to pH should be a stronger acid than the carboxylic acid to be recovered from the fermentation broth, preferably a strong mineral acid. A strong carboxylic acid can also be used. Acids having a pKa less than that of the carboxylic acid to be recovered can be used. The stronger the acid the lower the pKa value will be. The strong acid may shift the equilibrium of the carboxylic acid so that more of the carboxylic acid is in its protonated form. Examples of acids used to adjust the pH include, but are not limited to, arsenic, hydrobromic, hydrochloric, chloric, iodic, nitric, nitrous, phosphoric, phosphorous, hypophosphorous, pyrophosphoric, sulfuric, sulfurous, thiosulfuric, tellurous, formic, chloracetic, lactic, glycolic, citric and mixtures thereof.
The next step is to contact the fermentation broth with a suitable extraction solvent according to the present invention, i.e., a solvent containing one or more olefins. Olefins are well-known in the art. Examples such as cyclohexene (commercially available from Uniroyal Chemical Company, Inc., Naugatuck, Conn.), 1-hexene (commercially available from Chevron Chemical Company, Cedar Bayou, Tex.), 1-octene (commercially available from Shell Chemical Company, Geismar, La.), 1-decene (commercially available from Amoco Corporation, Pasadena, Tex.), diisobutylene, (commercially available from Texas Petrochemicals Corporation Houston, Tex.), Nonene (nonlinear) (propylene trimer) (tripropylene) (commercially available from Arco Products Co., Carson, Calif.), NEODENE(copyright) (commercially available from Shell Chemical Company, Houston, Tex.) and NEOSOLV(copyright) (commercially available from Shell Chemical Company, Houston, Tex.) perform as extraction solvents for the extraction of carboxylic acids from a fermentation broth in accordance with the present invention. Petroleum distillates containing one or more olefins are available such as kerosene, Lacolene (available from Ashland Distribution Company, Columbus, Ohio) and Varnish Makers and Painter""s (VMandP) Naptha (commercially available, e.g., from Ashland Distribution Company, Columbus, Ohio) are also suitable extraction solvents for the extraction of carboxylic acids from a fermentation broth. Thus, suitable olefins are aliphatic or alicyclic hydrocarbons with one or more double bonds along the chain. Higher olefins having chains of up to 20 or more carbon atoms and those that have a double bond between the first two carbons of the chain are known as alpha olefins, e.g., 1-decene or 1-dodecene.
At least one of the above described suitable extraction solvents are contacted with the fermentation broth for a time sufficient to extract carboxylic acids from the fermentation broth. Suitable time ranges are dependent upon the concentration of diacid in the broth, the viscosity of the broth, the temperature of the broth and other factors normally taken into account by those skilled in the art. Typical extraction times may range, e.g., from about 0.5 seconds or less to about 2 minutes or more. For example, the extraction time can be 15 minutes or more. The amount of extraction solvent may also be varied by those skilled in the art and can range from about {fraction (1/100)} of the broth volume to about 10 times the broth volume or more.
One or more co-solvents may be added to adjust the partition coefficient of the extraction solvent thus reducing the amount of solvent and/or stages of extraction required to sufficiently remove carboxylic acids from a fermentation broth. For example, the partition coefficient of diisobutylene in combination with the fermentation media and techniques described herein is typically less than about 1, i.e., about 0.5. As used herein, the partition coefficient is the concentration of diacid in extraction solvent by weight over the concentration of diacid by weight in the aqueous phase at equilibrium. In the case of carboxylic acids having 16 to 2.0 carbon atoms, the partition coefficient may be adjusted upward to a preferred range of about 3 to about 5 by addition of a suitable cosolvent which modifies the partition coefficient. Those skilled in the art will recognize that cosolvents which increase polarity are suitable for such adjustment. If the partition coefficient becomes too high, unwanted emulsion formation may occur as well as a loss in selectivity of the desired carboxylic acid. Routine experimentation by those skilled in the art may be utilized to determine the amount of cosolvent necessary to suitably adjust the partition coefficient. Suitable co-solvents include, but are not limited to esters of fatty acids and alcohols such as tert-butyl acetate and pentyl acetate, fatty alcohols such as 1-octanol, 2-octanol, dodecanol and decanol, aliphatic long chain ketones such as 2-octanone and 2-decanone, other water insoluble ketones such as methyl isobutyl ketone, ethers such as methyl tert-butyl ether, esters of fatty alcohols such as methyl hexanate, methyl octanate, ethyl hexanate and ethyl octanate and non-polar solvents such as chloroform, methyl chloride, toluene, benzene, xylene and the like.
A preferred extraction solvent for extraction of carboxylic acids from a fermentation broth is a mixture of tertiary-butyl acetate as a co-solvent and diisobutylene. Addition of tertiary butyl acetate to diisobutylene increases the partition coefficient of the solvent as compared to diisobutylene alone. In this aspect, the amount of diisobutylene may range from about 5% to about 99% and the amount of tertiary-butyl acetate may range from about 1% to about 95% by weight. Preferably, the solvent includes a mixture of a minority amount of tertiary butyl acetate and a majority amount of diisobutylene. A majority amount means that there is more diisobutylene than tertiary butyl acetate. In a preferred embodiment, the amount of diisobutylene is about 90% and the amount of tertiary-butyl acetate is about 10% by weight. Some advantages of using a solvent herein with a partition coefficient which has been adjusted upwardly as described above include the following: (1) less emulsion formation during extraction; (2) lower concentrations of carboxylate salts and sulfur are present in this solvent than when other solvents such as 2-octanol and 2-octanone are utilized alone; (3) these solvents are more environmentally friendly than other solvents such as for example, aromatic solvents; (4) diisobutylene and tertiary-butyl acetate have similar boiling points which allows easier recycling of the solvent without altering its composition; and (5) fewer repetitions of the extraction process are needed to obtain suitably pure product.
The extraction may be carried out using a continuous liquidxe2x80x94liquid extraction or may be accomplished batchwise. A preferred method is continuous liquidxe2x80x94liquid extraction. The batch or continuous extraction can be repeated numerous times to enhance recovery. If liquidxe2x80x94liquid extraction is performed in a batchwise manner, examples of systems include a stirred tank reactor, or a counter-current manner such as an extraction column or counter-currently configured CINC centrifugal extractor (commercially available from CINC, Carson City, Nev.). Preferably, the two phases should be contacted at a temperature greater than the melting point of the carboxylic acids such that the broth is fluid, preferably between 0xc2x0 C. and 100xc2x0 C., more preferably between 70-80xc2x0 C. and even more preferably about 75xc2x0 C. The two phases should be contacted for a length of time most preferably to allow the concentration of carboxylic acid in each phase to reach equilibrium. Once equilibrium has been established, mixing is ceased, the phases are allowed to separate, and the extraction solvent phase is separated from the broth phase by any method known to those skilled in the art. The solvent may then be separated from the carboxylic acids by any suitable method known to those skilled in the art, e.g., evaporation, distillation, melt crystallization, or crystallization. Crystallization may be effected by sufficient cooling of the extraction solvent phase to precipitate dicarboxylic acids followed by filtration of resulting crystals. The latter may also serve as a method of purification by selectively isolating carboxylic acids which crystallize at higher temperatures while allowing carboxylic acids which crystallize at lower temperatures to remain in the filtrate, which may then be isolated by evaporation of the solvent.
The isolated carboxylic acids can be further purified by an additional crystallization step or other means used by those skilled in the art such as distillation and chromatography methods. The recovered unsaturated carboxylic acids can be hydrogenated to remove double bonds. The unsaturated carboxylic acids can also be further reacted with an oxidizing agent to oxidatively cleave the carbonxe2x80x94carbon double bonds to carboxyl groups to form a polycarboxylic acid. The oxidative cleavage of the carbonxe2x80x94carbon double bonds may be achieved with any oxidizing agent known in the art which will oxidatively cleave a carbonxe2x80x94carbon double bond to form two carboxyl groups. Such methods include but are not limited to reaction with ozone and subsequent oxidative work-up of the ozonides as described in U.S. Pat. No. 2,813,113, the entire contents of which are incorporated herein by reference; reaction with tungstic acid in the presence of hydrogen peroxide, preferably 60% hydrogen peroxide as described in WO 94/10122, the entire contents of which are incorporated herein by reference; reaction with chromic acid as described in U.S. Pat. No. 2,450,858, the entire contents of which are incorporated herein by reference; reaction with hypochlorite in the presence of ruthenium oxide as described in J. Am. Oil Chem. Soc., 54, 870A (1977), the entire contents of which are incorporated herein by reference; permanganate oxidization as described in J. Am. Oil Chem. Soc., 54 (858A) (1977), the entire contents of which are incorporated herein by reference; peroxyformic acid oxidation as described in U.S. Pat. No. 5,380,928, the entire contents of which are incorporated herein by reference; cobalt bromide catalyzed peroxide oxidation as described in U.S. Pat. No. 4,606,863, the entire contents of which are incorporated herein by reference; cetylpyridinium chloride catalyzed phosphotungstic acid oxidation as described in JP 0183639, the contents of which are incorporated herein by reference.
In a particularly preferred embodiment the extraction of the carboxylic acids from the fermentation broth can be carried out without adjusting the pH of the broth using at least one of the above described extraction solvents.
Another preferred embodiment of the invention is based on the preparation of carboxylic acid by biooxidation of a crude mixture of oleic acid comprising a mixture of saturated and unsaturated carboxylic acids to form monocarboxylic and dicarboxylic acids, followed by extraction with a suitable extraction solvent as disclosed herein, recovery of the extracted carboxylic acids from the extractant by any conventional means, for example by recrystallization, followed by oxidation of the unsaturated carboxylic acid. While any crude mixture of oleic acid can be used, a particularly preferred crude mixture of oleic acid consists of the following % composition (GLC): 0.084 C12, 2.148 C14, 0.487 C14:1; 0.190 C15, 0.149 C15;1, 4.458 C16, 5.096 C16:1, 0.150 C17, 0.731 C18, 71.21 C18:1, 9.36 C18:2, 0.513 C18:3, 1.271 C20:1, and 0.330 C20:2.
Another preferred embodiment of the invention is based on the preparation of azelaic acid by biooxidation of oleic acid to form 9-octadecenedioic acid followed by oxidation of the 9-octadecenedioic acid to azelaic acid. While any grade of oleic acid can be used as the substrate, a typical technical grade oleic acid consists of the following carboxylic acids: 0.42% C12; 2.7% C14; 0.86% C14:1; 6.3% C16; 4.6% C16:1; 0.93% C17; 2.8% C18; 71.8% C18:1; 8.3% C18:2; 0.58% C18:3. The oleic acid can also be a high grade oleic acid obtained from a fatty oil of a Helianthus annuus (sunflower seed oil) species described, for example, in U.S. Pat. No. 4,627,192, the entire contents of which are incorporated herein by reference. Such oils are very rich in oleic acid and contain at least 80% by weight of oleic.
After the 9-octadecenedioic acid has been obtained by the biooxidation method disclosed herein, it can be recovered from the fermentation broth using the extraction method disclosed herein. pH adjustment of the broth is optional. The preferred pH range is from about 4.5 to about 5.5. The fermentation broth may be heated to about 65xc2x0 C. to about 75xc2x0 C. Heating the broth helps to reduce the viscosity to allow for better contact between the broth and the extractant. The broth is then contacted with the extraction solvent. The preferred means of contacting the broth with the extractant is via a continuous liquidxe2x80x94liquid extraction. The 9-octadecenedioic acid is recovered from the extraction solvent by any standard means as for example recrystallization.
After the 9-octadecenedioic acid has been obtained by the biooxidation and recovery methods disclosed herein, it may be reacted with ozone and further treated under oxidative conditions to yield azelaic acid. The mixed oxidation products are then further oxidized to azelaic acid as, for example, in the method disclosed in U.S. Pat. No. 5,420,316, the entire contents of which are incorporated herein by reference. A number of variations of the above azelaic acid preparation are contemplated for use with the present invention. For example, simple esters of oleic acid such as methyl oleate, ethyl oleate, and the like can be used in place of the oleic acid in the production of 9-octadecenedioic acid as well as natural fats and oils having a relatively high oleic acid content.
This process of using the extraction solvents described herein is superior when compared to techniques for isolating carboxylic acids from aqueous fermentation broth using saturated hydrocarbon solvents such as hexane, cyclohexane, heptane, and octane due to the increased solubility and partition coefficient of a containing solvent containing one or more olefins. This process is also superior to using solvents such as octanol, octanone, or other water insoluble ketones or alcohols. Although these alcohol and ketone solvents may have a greater extraction coefficient for the dicarboxylic acids, they also extract color containing impurities with the dicarboxylic acids, which are not seen in olefin extracted carboxylic acids according to the present invention. Alcohol and ketone solvents do not crystallize the dicarboxylic acids on cooling the separated extract as in olefin extracts but rather form a two phase system with an upper phase of solvent and a lower phase of carboxylic acids as residue phase that eventually becomes a solid with cooling. In addition, alcohol and ketone solvents have a greater solubility in water than do olefin based extractants according to the present invention. This greater water solubility leads to increased processing for removal of water from the extract and the removal of solvent from extracted broth. Carboxylic acids isolated by extraction using solvents according to the present invention contain less carboxylic salts or soaps than those isolated using alcohol or ketone solvents. Olefin based extraction solvents according to the present invention are also advantageous since they engender less emulsion formation during extraction than aromatic, alcohol and ketone solvents. The present extraction solvents are also superior to aromatic solvents such as toluene, xylenes, and other aromatic solvents since these aromatic solvents extract impurities leading to more color in the isolated diacids and are an environmental concern.
The following examples are meant to illustrate but not to limit the scope of the invention.